消旋聚乳酸复合rhBMP-2/hTGF-β1诱导小鼠成骨及其机制的研究

目的:评价消旋聚乳酸(PDLLA)载体材料复合重组rhBMP-2和hTGF-β1诱导小鼠体内异位成骨的能力,初步探讨其作用机制。方法:建立诱导RCI小鼠股部肌内异位成骨的18只动物模型,实验组(各6只)分别植入PDLLA/rhBMP-2/hTGF-β1和PDLLA/rhBMP-2复合材料,对照组(6只)植入PDLLA材料。采用图像处理技术、组织病理学和RT-PCR等分子生物学方法,对消旋聚乳酸载体材料复合2种细胞因子诱导小鼠体内异位成骨能力进行评价;并对其诱导异位成骨过程中,II型胶原蛋白、骨桥蛋白和骨钙蛋白等骨基质蛋白基因表达进行检测。结果:PDLLA/rhBMP-2/hTGF-β1和PDLLA/rhBMP-2组复合材料均具有明显的诱导小鼠肌内异位成骨能力,而单纯PDLLA载体材料无诱导异位成骨能力,表现为小鼠股部复合材料植入区的相对密度指数存在显著性差异。II胶原、骨桥蛋白和骨钙蛋白及其基因在2种细胞因子复合材料植入组中均有较高表达,而在单纯PDLLA组则无表达。结论:PDLLA复合rhBMP-2/hTGF-β1具有诱导异位成骨作用,软骨内成骨是其诱导小鼠体内异位成骨的主要方式。     

以胶原缓释rhBMP-2复合BMSCs及珊瑚构建组织工程骨异位成骨的实验研究

目的：观察以胶原缓释rhBMP-2复合BMSCs及珊瑚构建的组织工程骨异位成骨的能力。方法：构建3种复合支架材料：1)rhBMP-2／珊瑚；2)胶原rhBMP-2／珊瑚；3)BMSCs／胶原rhBMP-2／珊瑚。分别植入裸鼠皮下，8周后观察成骨情况，并作比较。结果：第3组材料异位成骨的能力最强，第2组次之，第1组较弱。结论：动物实验中胶原是rhBMP-2适宜的缓释载体，BMSCs对促进材料异位成骨有重要意义。     

山羊乳牙牙髓干细胞构建组织工程骨的异位成骨研究

目的:评价山羊乳牙牙髓干细胞在体外的成骨分化能力,及复合多孔磷酸钙骨水泥在山羊肌袋模型内的异位成骨能力。方法:采用改良组织块法培养山羊乳牙牙髓干细胞,并且在成骨诱导条件下培养。经成骨条件培养的第3代细胞与多孔磷酸钙骨水泥复合后,植入山羊背部左侧肌袋;将多孔磷酸钙骨水泥空白支架植入右侧肌袋作为对照组。分别在植入后4、8周取材,进行组织学观察。结果:复合山羊乳牙牙髓干细胞的支架材料在4周后有类骨质形成;植入8周后,类骨质形成更多;未复合细胞的支架材料在组织学检查中未见有类骨质形成。结论:山羊乳牙牙髓干细胞在体外具有分化为成骨细胞的能力;多孔磷酸钙复合山羊乳牙牙髓干细胞在山羊体内具有异位成骨能力。     

基因重组人骨形成蛋白—2与珊瑚／聚乳酸复合异位诱导成骨的实验研究

目的：观察基因重组人骨形成-2(rhBMP-2)和珊瑚／聚乳酸形成复合人工骨的异位诱导成骨活性。方法：把rhBMP-2和珊瑚／聚乳酸形成复合人工骨。进行小鼠肌内种植1，3，6周后，组织学观察其异位诱导成骨活性。结果：rhBMP-2赋予珊瑚／聚乳酸骨诱导能力，珊瑚／聚乳酸则充当rhBMP-2的载体和释放系统，对BMP的活性未不利影响。与单纯的珊瑚／聚乳酸相比，这种复合人工骨以软骨内成骨的方式诱导成骨。结论：rhBMP-2／珊瑚／聚乳酸复合人工骨具有良好的生物相容性和骨诱导活性，是一种较理想的骨移植替代材料。     

活性纳米羟基磷灰石复合胶原聚乳酸材料异位成骨性能研究

目的：探讨既具有骨引导性又具有骨诱导性的活性纳米羟基磷灰石复合胶原/聚乳酸（AnHAC/PLA）材料的异位成骨能力,为该材料作为植骨材料应用于临床打下实验基础。方法：通过扫描电镜观察rhBMP-2与nHAC/PLA材料的复合情况,并将nHAC/PLA、rhBMP-2、AnHAC/PLA材料分别植入小鼠股后肌袋内,术后2周、6周通过X线片、组织学观察、成骨量测定来研究AnHAC/PLA的异位成骨能力。结果：rhBMP-2在nHAC/PLA孔隙中均匀分布,6周时AnHAC/PLA组诱导形成的骨量是rhBMP-2组的10.5倍。结论：AnHAC/PLA材料具有良好成骨能力,nHAC/PLA是rhBMP-2理想的缓释载体。     

基因重组人骨形成蛋白-2和聚乳酸复合异位诱导成骨的实验研究

目的：观察基因重组人骨形成蛋白－2（recombinant human Bone Morphofenetic protein-2,rhBMP-2)和聚乳酸（Polylactic acid,PLA)复合形成的活性涂层种植体的异位诱导成骨活性。方法：将rhBMP-2与聚乳酸复合，构建活性涂层种植体，植入兔肌内，于1，4，8周后进行X线、大体观察及组织学观察，检测异位成骨活性。结果：rh-BMP-2/PLA活性复合涂层种植体具有骨诱导能力，PLA为rhBMP-2的良好控释载体。结论：rhBMP-2/PLA涂层具有良好的生物相容性和骨诱导活性，是一种理想的种植体涂层形成。     

基因重组人骨形成蛋白-2与珊瑚/聚乳酸复合修复骨质缺损的实验研究

目的　观察生物珊瑚、聚乳酸和rhBMP-2合成人工骨(复合骨)修复兔颅骨缺损后复合骨的变化情况。方法　选择24只新西兰兔,随机分成2组,每组12只,建立兔颅骨缺损标准模型。植入复合骨,用珊瑚/聚乳酸作为对照。术后4、8、12周每组各处死4只动物,取出植入体进行扫描电镜观察和机械强度测定。结果　复合骨在植入缺损后,不仅在植入体周边部有骨组织长入,而且在整个植入体内均有新骨形成,即出现多中心成骨。复合骨在同一时间点的成骨量明显多于对照组,随时间推移,成骨量递增。在植入前,两材料间的抗压强度无明显差异;在植入后,两植入体的抗压强度则差异显著,复合骨明显高于同期的对照组。结论　生物珊瑚、聚乳酸和rhBMP-2合成人工骨在体内以传导成骨和诱导成骨双重机制完成骨修复,且有良好的机械强度,作为植骨材料具有良好的应用前景。     

山羊乳牙牙髓干细胞在肌袋内的异位成骨

目的:评价山羊乳牙牙髓干细胞复合多孔磷酸钙骨水泥在山羊肌袋模型内的异位成骨能力.方法:采用改良组织块法培养山羊乳牙牙髓干细胞.经成骨诱导条件培养的第3代细胞与多孔磷酸钙骨水泥复合后,植入山羊背部左侧肌袋,将多孔磷酸钙骨水泥空白支架植入右侧肌袋作为对照组.分别在植入后2、4、6、8周取材,进行组织学观察,采用SPSS16.0软件包对数据进行统计学分析.结果:术后各时间点未复合细胞的pCPC支架材料组织学检查未见类骨质形成.SGDs-pCPC组中,术后2、4、6、8周成骨率分别为(1.24±0.25)％、(1.59±0.23)％、(4.12±0.39)％和(5.68±0.58)％.术后2周和4周之间的成骨率无显著差异(P＞0.05);术后8周的成骨率高于术后6周,且2者均显著高于2周和4周组的成骨率(P＜0.05).结论:多孔磷酸钙复合山羊乳牙牙髓干细胞在山羊体内具有异位成骨能力.     

rhBMP-2/Nha/Co复合膜异位成骨的实验研究

目的:观察rhBMP- 2/nHA/Co复合膜异位诱导成骨的能力.方法: 构建nHA/Co 复合膜和rhBMP- 2/nHA/Co复合生物膜,分别植入裸鼠后肢大腿肌囊,于10、20、30 d后观察成骨情况,并作比较.结果: rhBMP/nHA/Co组材料在裸鼠肌囊内出现骨样结构,异位成骨的能力较nHA/Co组强.结论: 动物实验中Co、nHA均为BMP的良好缓释载体,此复合膜能有效的诱导异位成骨.     

应用国产消旋聚乳酸载体复合rhBMP-2和hTGF-β1修复兔下颌骨缺损

目的评价国产消旋聚乳酸(PDLLA)载体材料复合重组人骨形成蛋白-2和转化生长因子-β1(rhBMP-2/hTGF-β1)整复兔下颌骨骨缺损的能力和载体材料的性能.方法建立健康成年家兔下颌骨骨缺损动物模型,分别应用PDLLA/rhBMP-2、PDLLA/rhBMP-2/hTGF-β1、PDLLA/hTGF-β1复合材料作为实验组,单纯PDLLA载体材料和空白组作为对照组,整复下颌骨缺损;通过大体解剖学观察、CT扫描和三维重建等影像学检查以及组织病理学检查等方法进行研究.结果术后2周时,各组下颌骨缺损区长度无差异;术后4周和8周时,PDLLA/rhBMP-2、PDLLA/rhBMP-2/hTGF-β1、PDLLA/haTGF-β1复合材料植入组,下颌骨缺损区长度与单纯PDLLA材料和空白组有显著性差异(P＜0.05),而复合材料各组间无差异.4～8周时,组织病理学检查显示实验组可见大量分泌旺盛的成骨细胞,并有明显的新骨形成,PDLLA材料逐步降解.结论国产消旋聚乳酸载体材料复合骨形成蛋白-2和(或)转化生长因子-β1具有促进骨缺损修复的作用,PDLLA可作良好的载体材料.     

Influence of bone morphogenetic protein and proportion of hydroxyapatite on new bone formation in biphasic calcium phosphate graft: Two pilot studies in animal bony defect model

PurposeThe purpose of these two pilot studies using animal bony defect models was to evaluate the influence of bone morphogenetic protein (BMP) and proportion of hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) in biphasic calcium phosphate (BCP) graft on new bone formation.MethodsIn this study, four kinds of synthetic osteoconductive bone materials known for bone growth scaffold, OSTEON™II(HA:β-TCP 30:70), OSTEON™III (HA:β-TCP 20:80), OSTEON™II Collagen, and OSTEON™III Collagen, were prepared as BCP graft materials. In pilot study 1, three BCP materials (OSTEON™II, OSTEON™III, and OSTEON™II Collagen) were grafted in rabbit calvarial defects after impregnating in rhBMP-2. OSTEON™II without the rhBMP-2 impregnation was included in the study as the control. The amount of new bone was examined and measured histologically at 2, 4, and 8 weeks. In pilot study 2, four BCP materials (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) were grafted in beagle dog mandibular defects after soaking in the rhBMP-2. The amount of total bone and new bone were measured three-dimensionally using microCT and healing process was examined histologically at 2, 4, and 8 weeks.ResultsIn pilot study 1, rhBMP-2 impregnated groups showed more new bone formation than the rhBMP-2 free group. In pilot study 2, increased new bone formation was observed in time-dependent manner after graft of BCP and BCP-collagen (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) impregnated with rhBMP-2. Also, BCP with a higher proportion of HA (30% HA) showed more favorable result in new bone formation and space maintenance, especially at the 8 weeks.ConclusionFrom the results of the pilot studies, rhBMP-2 played positive roles in new bone formation and BCP could become a scaffold candidate for rhBMP-2 impregnation to induce new bone formation. Moreover, BCP with a higher proportion of HA (30% HA) could be considered more appropriate for rhBMP-2 carrier.     

三维打印β-TCP颌骨修复支架的生物学评价

目的:探讨三维打印β-磷酸三钙(β-Tricalcium Phosphate, β-TCP)颌骨修复支架的生物学特性及体内成骨作用。方法:采用自动注浆技术制作β-TCP支架,将前成骨细胞(MC35T3-E1)接种在支架上,扫描电镜(SEM)观察材料结构与细胞黏附,CCK-8法检测细胞增殖,ALP法检测碱性磷酸酶活性。将2种支架复合重组人骨形成蛋白-2(recombinant human bone morphogenetic protein-2, rhBMP-2)后植入大鼠体内,发泡法制作的β-TCP支架为对照组,6周后取材行组织学观察。结果:三维打印支架具有规则多孔的立体结构,适合细胞黏附,且增殖及分化能力均高于对照组(P<0.05)。组织学显示复合rhBMP-2后三维打印支架新骨生成量高于发泡法制作的β-TCP支架(P<0.05)。结论:三维打印TCP支架生物相容性良好,复合rhBMP-2后可异位成骨。     

Bone regeneration of critical calvarial defect in goat model by PLGA/TCP/rhBMP-2 scaffolds prepared by low-temperature rapid-prototyping technology

Active artificial bone composed of poly lactide-co-glycolide (PLGA)/ tricalcium phosphate (TCP) was prefabricated using low-temperature rapid-prototyping technology so that the process of osteogenesis could be observed in it. PLGA and TCP were the primary materials, they were molded at low temperature, then recombinant human bone morphogenetic protein-2 (rhBMP-2) was added to form an active artificial bone. Goats with standard cranial defects were randomly divided into experimental (implants with rhBMP-2 added) and control (implants without rhBMP-2) groups, and osteogenesis was observed and evaluated by imaging and biomechanical and histological examinations. The PLGA-TCP artificial bone scaffold (90% porosity) had large and small pores of approximately 360microm and 3-5microm diameter. Preliminary and complete repair of the cranial defect in the goats occurred 12 and 24 weeks after surgery, respectively. The three-point bending strength of the repaired defects attained that of the normal cranium. In conclusion, low-temperature rapid-prototyping technology can preserve the biological activity of this scaffold material. The scaffold has a good three-dimensional structure and it becomes an active artificial bone after loading with rhBMP-2 with a modest degradation rate and excellent osteogenesis in the goat.     

Bone engineering of the rabbit ulna.

PURPOSE: The purpose of the present preliminary study is to show that a novel 3-dimensional porous silica-calcium phosphate nanocomposite (SCPC) can provide a controlled release of rhBMP-2 and regenerate bone in a load-bearing segmental defect. MATERIALS AND METHODS: A bone replica of the rabbit ulna was created from SCPC powder using rapid prototyping technology. The ceramic bone replica was coated with rhBMP-2 and then implanted into a 10-mm segmental defect created in a rabbit ulna and fixated with a 1-mm titanium adaptation plate. Bone healing was evaluated using computed tomography (CT) scan, histomorphometry, and biomechanical techniques. The release kinetics of rhBMP-2 and the dissolution kinetics were also determined in vitro. Statistical analysis was performed to compare the biomechanical strength of the grafted bone with the contralateral unoperated ulna. RESULTS: After 4 weeks, CT scans showed that the critical size defect had been replaced by newly formed bone. Torsional testing of the ulna after 12 weeks showed restoration of maximum torque and angle at failure. Histological evaluation showed that the regenerated bone had the morphological characteristics of mature bone. SCPC provided a sustained release profile of an effective dose of rhBMP-2 for 14 days. CONCLUSIONS: The SCPC-rhBMP-2 hybrid enhanced bone regeneration in a load-bearing segmental defect in a rabbit ulna. The regenerated bone acquired morphology and mechanical strength typical for natural bone. The enhanced bone formation correlates well with the surface bioactivity and effective release profile of rhBMP-2. The present preliminary study shows the proof of principles that porous, resorbable, bioactive SCPC-rhBMP-2 tissue engineering hybrid can serve as a substitute for autologous bone in load-bearing applications.     

Enhancement by recombinant human bone morphogenetic protein-2 of bone formation by means of porous hydroxyapatite in mandibular bone defects.

Hydroxyapatite is osteoconductive and can maintain an original biocompatible form. It is useful, in the reconstruction of bone defects, to enhance the osteoconduction of hydroxyapatite with an osteogenic protein. The aim of this study was to evaluate the bone formation in surgically created defects of rabbit mandibles by a combination of recombinant human bone morphogenetic protein-2 (rhBMP-2), with porous hydroxyapatite and atelopeptide type I collagen used as the carrier for rhBMP-2. A 10-microg rhBMP-2-implanted group (n = 15) and a control group (n = 15), in which only atelopeptide type I collagen and porous hydroxyapatite were implanted, were histologically examined 3, 7, and 21 days after implantation. The alkaline phosphatase activity was also quantitatively analyzed. No new bone formation was observed in either the tested or the control group after 3 days. At 7 days, immature bone tissue was observed in some pores of the rhBMP-2implanted group, while in the control group, immature mesenchymal cells were observed. At 21 days, trabecular bone lined some pore walls. In the central portion, the bone marrow, including angioid tissue, was observed. New trabecular bone formation was observed on portions of the external surface of the hydroxyapatite disk. On the other hand, the control group showed infiltration of immature mesenchymal cells into some pores. Marginal bone formation was found in the pores close to the surface of the disk which opposed mandibular bone. The control group showed a slow, small increase in alkaline phosphatase activity in this study, while the experimental group showed a marked increase at 21 days. This increase was significantly higher in the tested group than in the control group at both 7 and 21 days. The findings indicate that rhBMP-2 accelerated bone formation by osteoconduction from porous hydroxyapatite. The combination of rhBMP-2, atelopeptide type I collagen, and porous hydroxyapatite is suggested to be advantageous for clinical application in reconstructing mandibular bone defects.     

Bone with a vascular flap induced from fat tissue with the use of rhBMP-2 in rats

Here we report that successful bone formation with a vascular flap inside a cylindrical mold was induced from fat tissue with the use of recombinant human bone morphogenetic protein-2 in rats. Fat tissue connected to blood vessels was prepared to fit into the mold and implanted intramuscularly into the hind leg in Wistar rats. RhBMP-2 (20 micro g) was applied in a collagen sheet previously placed on the inside surface of the mold. Bone formation was confirmed radiologically and morphologically at 2, 4, and 8 weeks after the surgery. In the control group without rhBMP-2 or the group with ligation of the blood vessels before the implantation, bone formation was not observed. Our success in bone formation having a definite size, shape, and blood supply may lead to a therapeutic approach to effective bone reconstitution. The present study is the first report on bone induction from fat tissue by rhBMP-2 in vivo.     

重组人骨形成蛋白-2促进兔下颌牵张成骨的研究

目的　研究局部应用基因重组人骨形成蛋白-2(rhBMP-2)对兔下颌牵张成骨的影响。方法　在12只成年大耳白兔的双侧下颌骨前部行骨切开术,将rhBMP-2与胶原复合植入一侧下颌骨切开处,另一侧单纯植入胶原作对照。用自行研制牵张器延长双侧下颌骨6 mm,在牵张结束后第4周处死动物,取双侧牵张区新生骨痂行组织学、扫描电镜及Ca/P元素测定。结果　下颌延长后两侧牵张间隙均有新骨形成,应用rhBMP-2的一侧牵张骨痂中的新骨组织比对照侧多而成熟,钙化程度较高。结论　基因重组人骨形成蛋白-2可能有促进兔下颌牵张成骨的作用。     

新型骨支架复合材料修复牙槽骨缺损研究

目的:探讨多孔聚丙烯晴基碳纤维/壳聚糖/双相磷酸钙/聚乳酸-羟基乙酸(PAN/CS/BCP/PLGA)复合支架对大白兔牙槽骨缺损的修复能力。方法:采用真空冷冻干燥技术制备多孔PAN/CS/BCP/PLGA复合支架。选取32只成年雄性新西兰大白兔,随机分为实验组和对照组,手术制备大小为10mm×10mm×10mm牙槽骨缺损区。实验组骨缺损区植入PAN/CS/BCP/PLGA复合支架,对照组植入CS/PLGA复合材料,术后2、4、8、12周分期取材,进行X线检查、组织学观察、扫描电镜(SEM)检测和成骨细胞计数分析。结果:术后12周实验组检测结果,X线观察:骨缺损区阻射影与自体骨相同。组织学观察:骨缺损区完全被新骨充填,且为较成熟的板层骨,与自体骨完全融合。SEM检测:骨-材料界面形成骨性结合,移植材料大部分降解,被成熟骨组织替代。成骨细胞计数分析:成骨细胞生长活跃,统计学有显著性差异。结论:多孔PAN/CS/BCP/PLGA复合支架具备良好的生物活性和生物可降解性,对骨缺损的修复能力要优于CS/PLGA复合材料,为其成为骨组织工程支架替代材料提供理论依据。     

Bone gap healing in the dog using recombinant human bone morphogenetic protein-2.

PURPOSE: This study investigated bone gap healing in a zygomatic arch defect using recombinant human bone morphogenetic protein-2 (rhBMP-2; Genetics Institute, Andover, MA) in an absorbable collagen sponge (ACS) carrier. METHODS: Zygomatic arch osteotomies were completed 15 mm apart and the arch was mobilized in 6 adult female mongrel dogs. The segment was then repositioned laterally 8 to 10 mm and secured with a titanium reconstruction plate. Bone gaps in either the right or left arches received rhBMP-2, with the contralateral side being left empty in 4 animals and the defects received buffer/ACS without rhBMP-2 in 2 animals as controls. Submentovertex radiographs were taken immediately postoperatively and every 4 weeks until killing at 12 weeks. RESULTS: Clinical evaluation indicated no significant differences in the degree of inflammation between the groups. However, the rhBMP-2 sites were found to be firm on palpation, in contrast to a soft tissue defect palpated in the control sites. Radiographic examination showed significant bone formation in all rhBMP-2 grafted sites as early as 4 weeks. The radiopacity of the bone continued to increase over the time of this study. Five of six control sites did not show bone formation through the course of this study. In addition to lack of bone formation, 5 of 6 control sites showed collapse of the repositioned arch. All arches in the rhBMP-2 sites remained in their lateral position and formed bone in the gaps. In 2 animals, bone formation moderately exceeded the confines of the gap, and in 2 animals excessive bone formation occurred. CONCLUSIONS: This study confirms that rhBMP-2 has the potential to be used to stimulate bone gap healing in the craniofacial complex.